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N-Glycan Modification in Aspergillus Species

Elke Kainz,^1, Andreas Gallmetzer,^1, Christian Hatzl,^1, Juergen H. Nett,² Huijuan Li,² Thorsten Schinko,¹ Robert Pachlinger,^1, Harald Berger,¹ Yazmid Reyes-Dominguez,¹ Andreas Bernreiter,¹ Tillmann Gerngross,^2,3 Stefan Wildt,² and Joseph Strauss^1*

Fungal Genomics Unit, Austrian Research Centers and BOKU Vienna, Muthgasse 18, A-1190 Vienna, Austria,¹ GlycoFi, Inc., 21 Lafayette Street, Lebanon, New Hampshire 03766,² Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, New Hampshire 03755³

Received 11 May 2007/ Accepted 2 December 2007

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional -1,2-mannosidase producing increased amounts of N-glycans of the Man₅GlcNAc₂ type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan₅GlcNac₂ N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man₃GlcNAc₂ N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi.

Published ahead of print on 14 December 2007.

E.K. and A.G. contributed equally to this work.

Present address: Octapharma Produktionsges. m.b.H., Oberlaaerstrasse 235, A-1100 Vienna, Austria.

Present address: Baxter AG, Industriestrasse 67, A-1221 Vienna, Austria.